Molecular cloning and characterization of soybean peroxidase gene families

Huabang Chen, Purdue University

Abstract

Peroxidases are a ubiquitous class of enzymes and play major roles in many plant physiological processes. The objectives of this study were to isolate and characterize soybean peroxidase cDNA and genomic DNA, and to study in vitro folding conditions of bacterially expressed soybean peroxidase. Five full-length soybean peroxidase cDNA, GmEPa1, GmEPa2, GmEPb1, GmEPb2 and GmEPc, were isolated from a soybean seedbud cDNA library (Glycine max. Resnik). Neutral peroxidases, GmEPa1 and GmEPa2 were expressed in developing seed and root, the cationic peroxidases GmEPb1 and GmEPb2 were detected in root, stem, leaf, and seedpod, and the anionic peroxidase GmEPc was previously shown to be expressed in seed coat and root. These different peroxidases may play different roles in soybean growth, development, and/or defense system against pathogen attacks. The availability of these genes and gene-specific primers are essential for further in vivo and in vitro studies of their expressions, functions and catalytic mechanisms. Three soybean peroxidase genomic DNA fragments, GmEPA1, GmEPB1 , and GmEPC, were isolated from a soybean genomic library and correspond to cDNA GmEPa1, GmEPb1, and GmEPc, respectively. Genomic Southern blot analysis indicated that a very small number of these three genes were present in the soybean genome. The 1391 bp 5′ flanking region of GmEPA1 was capable of conferring both seed coat and root expression of GUS by transient expression assay. The 1524 bp promoter of GmEPC was capable of driving the expression of GUS in the seed coat. This promoter and regulatory sequences could be used to drive expression of proteins of industrial and human interest in soybean seed coats. The open reading frame including the 5′ leader sequence of GmEPa1 was cloned into the pET-34(+) expression vector. This peroxidase enzyme was expressed as a CBD-peroxidase fusion protein in inclusion bodies. The recovery of peroxidase activity was critically dependent on the addition of hemin and on the concentration of urea, with ∼2M being optimal at pH 8.0. The best overall in vitro folding efficiency (∼5%) was achieved when 50% ethylene glycol was included in the standard folding buffer. The folding efficiency was high enough for mutagenesis studies.

Degree

Ph.D.

Advisors

Vierling, Purdue University.

Subject Area

Molecular biology|Biochemistry

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